During the initial tenure of this Program Project, studies have provided new insights into the trafficking and function of the vasopressin-sensitive water channel, the role of the cytoskeleton in the modulation of cell structure and function, the regulation of vesicle endo- and exocytosis and the generation of functional polarity in kidney epithelia, and the role of Ca++ and phospholipids in signal transduction. Taking advantage of novel tools and cell systems that we have developed over the past four years, Project 7 will now characterize the specialized early endosomal vesicles that internalize vasopressin-induced water channels, define the cell biology of their recycling, and eventually isolate the water channel itself. Project 8 will utilize a unique melanoma cell line that is deficient in actin binding protein (ABP), fails to modulate ion channels and cannot volume-regulate. These functions can be totally restored by transfecting the cells with ABP cDNA, and these cells provide a powerful system in which to examine interactions between the cytoskeleton and membrane function that are relevant to renal epithelia. Project 9 will investigate the regulation of epithelial secretory and membrane protein trafficking in the Golgi. It will take advantage of our newly-developed renal cell lines transfected with inducible genes for heterotrimeric GTP- binding protein alpha subunits to pursue our novel discovery that these proteins work in concert with monomeric G-proteins to regulate Golgi traffic. Project 10 will examine the role of microtubules and their associated proteins, MAPs, involved in the generation of the polarized epithelial cell phenotype. It will define the requirement for cross-talk between microtubule- and actin-regulatory proteins in this process. Project 11 will define the intracellular sorting and polarized targeting of MHC I. This project take advantage of hybrid MHC constructs, transfected kidney epithelial cell lines, and domain and conformation-specific antibodies to investigate the contribution of a "passenger" protein in vesicle trafficking. Finally, Project 6 will continue to elucidate Ca++ and phospholipid metabolism as they relate to hormonal signal transduction and cell proliferation in glomerular mesangial cells. Emphasis will be placed on the important and previously unappreciated role of actin- regulatory proteins in phospholipid metabolism. As in the past, this Program Project brings together a group of scientists with considerable expertise in many aspects of cell biology, molecular biology, biophysics, biochemistry and morphology which will enable us to examine in greater depth interactions between the plasma membrane and the cytoplasm of renal cells that are important both in normal function and in disease.